The objective of this proposal is to identify leptospiral outer membrane proteins (OMPs) that have the capacity to serve as protective immunogens. Leptospirosis is an important, global human and veterinary health problem. Commercially available whole cell vaccines are highly toxic, produce only short-term immunity, and provide little cross- protection against infection with many of the 170 different leptospiral serovars. Whole cell vaccines have been shown to be ineffective in protection of cattle from infection by serovar hardjo, a prevalent North American leptospiral serovar. For these reasons, there is a critical need for development of new vaccine strategies for prevention of leptospirosis. The genes encoding four surface-exposed leptospiral OMPs have been cloned and sequenced: OmpL1, OmpL2, OmpL36, and LipL41.
Specific Aim #1 is to evaluate the capacity of recombinant OMPs, alone or in combination, to serve as protective immunogens. Two complementary animal models will be used: fulminant leptospirosis in hamsters, and sublethal infection of BALB/c mice resulting in the carrier state. Animals will be challenged with virulent Leptospira by two routes: intraperitoneal and conjunctival inoculation. Sublethal infection will be detected by four methods: culture, antibody response, immunohistochemistry, and by the polymerase chain reaction. We have developed a novel method of purifying the leptospiral outer membrane in the form of unilamellar vesicles. The outer membrane vesicles contain four additional OMPs with molecular masses of 20-, 46-, 51-,, and 59-kDa.
Specific Aim #2 is to clone and sequence their genes, express them as recombinant proteins, generate specific antisera, and determine whether they have surface-exposed epitopes by surface immunoprecipitation and immunoelectron microscopy.
Specific Aim #3 is to determine whether protective immunogens are expressed during infection by evaluating the humoral immune response of hamsters infected with host-adapted Leptospira, and by immunohistochemistry of Leptospira found in host tissues during infection.
Specific Aim #4 is to study how OMPs serve as protective immunogens. Passive immunization will be performed to assess the contribution of humoral immunity. The ability of immune serum and complement to kill Leptospira will be used to determine if OMPs are targets of bactericidal antibody. Immune serum will also be used to block leptospiral adherence to eucaryotic cells in tissue culture. It is anticipated that these studies will improve the diagnosis and prevention of leptospirosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI034431-02
Application #
2413643
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1996-05-01
Project End
2001-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Witchell, Timothy D; Eshghi, Azad; Nally, Jarlath E et al. (2014) Post-translational modification of LipL32 during Leptospira interrogans infection. PLoS Negl Trop Dis 8:e3280
Matsunaga, James; Schlax, Paula J; Haake, David A (2013) Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans. J Bacteriol 195:5092-101
Lambert, Ambroise; Picardeau, Mathieu; Haake, David A et al. (2012) FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath. Infect Immun 80:2019-25
Verma, Ashutosh; Matsunaga, James; Artiushin, Sergey et al. (2012) Antibodies to a novel leptospiral protein, LruC, in the eye fluids and sera of horses with Leptospira-associated uveitis. Clin Vaccine Immunol 19:452-6
Narayanavari, Suneel A; Sritharan, Manjula; Haake, David A et al. (2012) Multiple leptospiral sphingomyelinases (or are there?). Microbiology 158:1137-46
Pinne, Marija; Matsunaga, James; Haake, David A (2012) Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins. J Bacteriol 194:6074-87
Choy, Henry A (2012) Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement. PLoS One 7:e41566
Eshghi, Azad; Pinne, Marija; Haake, David A et al. (2012) Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32. Microbiology 158:622-35
Pinne, Marija; Haake, David (2011) Immuno-fluorescence assay of leptospiral surface-exposed proteins. J Vis Exp :
Coutinho, Mariana L; Choy, Henry A; Kelley, Melissa M et al. (2011) A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans. PLoS Negl Trop Dis 5:e1422

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